What Makes each Aux/IAA Gene Unique in its Gene Family, Expression Pattern or Properties of the Gene Product?

Abstract

In the auxin signal transduction, two protein families, Aux/IAAs and auxin response factors, play a crucial role just downstream of auxin F-box receptors. Distinct and overlapping phenotypes of the dominant Aux/IAA mutants suggest some functional differentiation of the Aux/IAA genes in auxin signaling. Taking advantage of unique phenotypes of the msg2/iaa19 mutants, we carried out promoter-exchange experiments, where cDNA of the msg2, axr2/iaa7 or slr/iaa14 gene was driven by the MSG2 or AXR2 promoter. The cDNAs were translationally fused to the green fluorescent protein gene to measure levels of expressed protein. Results showed that many abnormal phenotypes of the dominant Aux/IAA mutants were governed by their promoter activity, but some were dependent on their gene products. The latter result highlights the possible importance of Aux/IAA protein level controled by auxin F-box receptors.

Auxin exerts many physiological responses in different tissues, which has been a puzzle since its discovery in the 1920's. The discovery of two protein families, auxin response factors (ARFs) and Aux/IAAs, in the late 1990's was, thus, epoch-making because each physiological response might result from combinatorial interaction between a subset of the ARF and Aux/IAA families, which consist of 23 and 29 proteins in Arabidopsis, respectively.^1,2 Consequently, each Aux/IAA gene is thought to be differentiated in their physiological function, at least to some extent. Consistent with this idea, dominant Arabidopsis mutants of the Aux/IAA genes show both distinct and overlapping phenotypes. The next question is: What makes each Aux/IAA differentiated on a molecular level?

Theoretically there would be two extremes for this question. One is that the function of Aux/IAAs is solely decided by their expression pattern. In this case, all the dominantly mutated Aux/IAA proteins (mAux/IAAs) would produce similar defects if expressed in the same tissue. The other is that each Aux/IAA could interact with a distinct set of ARFs, leading to phenotypic defects characteristic to the repressed ARFs. In this case, each mAux/IAAs should induce qualitatively different defects even if expressed by the same promoter. This question has been addressed before by Knox et al.³ and Weijers et al.⁴ by the use of the promoter-swapping strategy. In the latest issue of Plant Physiology we also reported our results on this question⁵ by taking advantage of the msg2/iaa¹⁹ mutants, which exhibit fewer defects than the other dominant Aux/IAA mutants.^6,7 Figure 1 summarizes our results as well as those of Weijers et al.⁴ In our experiments, cDNA of msg2, axr2/iss7⁸ or slr-1/iaa14⁹ was driven by the MSG2 or AXR2 promoter. Weijers et al. expressed the bdl/iaa12¹⁰ or shy2/iaa3¹¹ cDNA by the BDL or SHY2 promoters.

Figure 1.

Promoter- and gene product-dependent phenotypes of the dominant Aux/IAA mutants as revealed by promoter-exchange experiments. pBDL represents promoter of the BDL gene.

Of the 21 determined phenotypes in total, mAux/IAA proteins induce the same or qualitatively similar defects in 17 phenotypes (Fig. 1). In four cases, however, mAux/IAA did exert qualitatively different phenotypes, even when driven by the same promoter. This clearly shows that physiological function of mAux/IAA was determined by both the pattern of gene expression and the properties of gene products, but that gene expression may have a primary role. This conclusion is essentially the same as that reached by the previous study.⁴

The importance of gene expression has been widely recognized in studies of gene function. Thus, it would be surprising if each mAux/IAA protein had distinct characteristics, and the next question would be: What properties of the Aux/IAA proteins make them distinct from each other? The Aux/IAA proteins consist of three conserved regions, domain I, domain II and the carboxy-terminal domain (CTD). Domain II is a recognition site for auxin F-box receptors (AFBs).^12–14 AFBs ubiquitinate Aux/IAAs after auxin perception, leading to degradation of Aux/IAA. This relieves ARFs from repression of their transcriptional activities, which ultimately results in auxin responses. Therefore, properties of Aux/IAAs are likely determined by the binding constants for ARFs through CTD and for AFBs through domain II. Because strength of interaction between Aux/IAAs and ARFs appears to be similar for pairs investigated so far with yeast two-hybrid assay^{4,6,9,10,15,16} or fluorescence cross-correlation spectroscopy,¹⁷ binding constants between Aux/IAAs and AFBs may be variable. In fact, when driven by the same AXR2 or MSG2 promoter, a protein level of msg2-1 estimated from fluorescence intensity of green fluorescent protein fused to msg2-1 was much lower than that of axr2-1,⁵ suggesting that msg2-1 has a higher affinity to AFBs. This difference may cause a few msg2-specific defects independent of the promoter activities (Table 1, underlined). Even in the case where mAux/IAAs exhibited quantitatively different phenotypes, msg2-1 exerted weaker defects than did slr-1 and axr2-1 (Table 1, shaded). This may also be due to a lower msg2 level than the other two mAux/IAAs. Quantitative determination of the interaction between Aux/IAAs and AFBs will be needed to further understand functional differentiation of the Aux/IAA family.

Table 1.

Shared and differentiated functions among the dominantly mutated Aux/IAA proteins

Phenotype	Degree of Defects					Target Auxin Response Factors (ARF) (Putative)
	Dominant Mutants	Driven by MSG2 Promoter		Driven by AXR2 Promoter
Size of nature plants	wt1) = msg2 < slr < axr2	wt =	msg2 = slr = axr2*	wt =	msg2 < slr = axr2	ARF6 + ARF818
Embryogenesis	wt = msg2 = slr = axr2 +NPH4/ARF716	wt =	msg2 < slr = axr2	wt =	msg2 = slr = axr2*	MP/ARF5
Shape of etiolated hypocotyls	wt ≈ msg2 < slr, axr22)	wt ≈	msg2 = slr = axr2	wt ≈	msg2 < slr, axr22)	ARF6 + ARF818
Hypocotyl gravitropism	wt < msg2 < slr < axr2	wt <	msg2< slr = axr2	wt <	msg2< slr = axr2	NPH4 + ARF1919,20
Lateral root formation	wt < msg2, axr22) < slr	wt <	msg2 = slr = axr2	ND1)		NPH4 + ARF1920,21
Root gravitropism	wt = msg2 < slr = axr2	wt =	msg2 = slr = axr2*	ND		NPH4 + ARF1920
Root hair formation	wt = msg2 < slr = axr2	wt =	msg2 = slr = axr2*	ND		?

^*This shows that MSG2 or AXR2 does not express in a tissue critical for the phenotype. Proteins appearing in a black background show the same function among the mutated Aux/IAA proteins; those in a grey background show quantitative differences in function among them; underlined names show qualitative differences;

¹⁾wt, wild type; ND, not determined;

²⁾Two mutants exhibit qualitatively different phenotypes.

Addendum to: Muto H, Watahiki MK, Nakamoto D, Kinjo M, Yamamoto KT. Specificity and Similarity of Functions of the Aux/IAA Genes in Auxin Signaling of Arabidopsis Revealed by Promoter-Exchange Experiments Between MSG2/IAA19, AXR2/IAA3 and SLR/IAA14. Plant Physiol. 2007;144:187–196. doi: 10.1104/pp.107.096628.